PROJECT SUMMARY Chronic metabolic dysfunction has emerged as one of the most severe medical problems worldwide, leading to increases in type 2 diabetes, insulin resistance, and cardiovascular disease. The discovery of alternative pathways to regulate whole-body glucose and energy metabolism is urgently needed to address this great medical need. Such pathways could be exploited for new therapeutic strategies to combat diabetes and insulin resistance. Using a multidisciplinary strategy combining computational, cellular, and in vivo approaches, we have recently uncovered a new adipokine from thermogenic adipose, Isthmin-1 (Ism1), that acts to promote glucose uptake in mouse and human adipocytes. The action of Ism1 requires PI3K/AKT signaling but is entirely independent of the insulin receptor. In animals rendered diabetic by high-fat diet feeding, administration of recombinant Ism1 protein or genetic elevation of circulating Ism1 improves glucose homeostasis. However, more studies are needed in order to understand the contribution of Ism1 to glucose metabolism, and to leverage this understanding for therapeutic purposes. The overall objectives in this proposal are to establish how Ism1 can control blood glucose by determining the signaling effectors and cell surface receptor that mediate the action, determine the endogenous physiological function for Ism1, and evaluate the pharmacological potential of Ism1 as a therapeutic target. In Aim 1, we will utilize biochemical, genetic, and proteomic methods to identify the signaling pathways and cell surface receptor responsible for the signaling action and glucoregulatory mechanisms of Ism1. These studies will identify Ism1?s mechanism of action and will be critical for our understanding of Ism1 signaling as an insulin-independent pathway to regulate glucose uptake. In Aim 2, we will determine the physiological function for Ism1 using our generated whole-body and adipocyte-specific Ism1 knockout mice. These studies are essential in determining the endogenous role of Ism1 in glucose metabolism. In Aim 3, we will determine the minimal requirements for Ism1 bioactivity by generating fragments, mutants, and engineered forms of Ism1. This aim will pave the way for further optimization of a polypeptide hormone as a therapeutic agent, and will be essential in understanding the effects of augmentation of this novel pathway physiology. These contributions are expected to be significant because pathways that can regulate glucose independently of insulin will open entirely new avenues to overcome insulin resistance and diabetes, which could have a significant public health impact.